A problem that exists in the field of culturing or proliferating stem cells, progenitors, induced pluripotent stem cells or other non-adherent cells, is how to culture the cells in a way that will not interfere with later intended uses, including transplants or downstream differentiation. Unlike most cells that adhere to plastic, which can be cultured in plastic growth flasks, stem cells are non-adherent, therefore cannot be grown using traditional methods. Stem cells will, however, grow on layers of fibroblast cells. These “feeder cell” layers provide a surface for adhesion and feed the cells with a mixture of as yet uncharacterized growth factors that are required for stem cell growth and survival. More recently, researchers have been able to culture stem cells by attaching them to components derived from the extracellular matrix, such as matrigel. Stem cells adhere to these surface-like substrates but must be cultured in growth media that contains both basic fibroblast growth factor (bFGF) and collected secretions from fibroblast feeder cells. It is not entirely clear how or why these methods promote stem cell proliferation, since they both use a milieu of uncharacterized factors secreted by cells. It has been reported that stem cells differentiate more quickly when they are cultured over matrigel surfaces. Stem cells grown according to either method, i.e. feeder cells or matrigel plus conditioned media from feeder cells, spontaneously differentiate. Differentiating stem cells secrete factors that induce neighboring cells to also initiate differentiation. Therefore, every approximately 7 days, a technician must manually dissect and harvest only those stem cell colonies or colony portions that appear to be undifferentiated. The harvested cells are then re-plated for continued growth. This procedure is repeated until enough undifferentiated cells can be harvested for the intended purpose. These methods for culturing stem cells are the state of the art for the industry.
Virtually any kind of scaled up growth of stem cells or induced pluripotent stem cells will require the development of new methods that enable high throughput harvesting of these cells.
The current practice is to grow stem cells on fibroblast feeder cells from which the only method of harvesting is by manual dissection, under a microscope, and isolation of “good” cells, followed by re-plating. This procedure is flawed because it is subjective and time consuming. What is needed are methods for automatable harvesting and automatable methods for purifying the desired cells from a mixed pool wherein cells are selected on the basis of molecular recognition rather than subjective criteria of a technician.
The state of the art methods for culturing stem cells are inadequate because they: 1) are labor-intensive; 2) are inherently incompatible with large scale growth; 3) depend on uncharacterized factors such as conditioned media; 4) require cells or cellular products for adhering stem cells to growth flasks; and 5) frequently use non-human cells and cellular extracts that can irreversibly change the human stem cells. A significant improvement would be if discrete factors that enable stem cell growth were identified. It is thought that if only the necessary and sufficient growth factors were added, then spontaneous differentiation would be minimized. Another significant improvement would be if cells could be safely harvested in a manner that was compatible with large scale growth rather than the current method of manual dissection under a microscope. Currently, stem cells growing on matrigel can be harvested by enzymatic cleavage, e.g. using trypsin. Typically, undifferentiated colonies or portions of colonies are manually dissected then digested with an enzyme such as trypsin or collagenase. However, trypsin causes significant cell death and serial passaging of stem cells on matrigel has been reported to cause abnormal karyotype. This could be due to harvesting with trypsin or may be due to the fact that matrigel is a mixture of cells and secretions from mouse sarcoma cells. Non-human feeder cells have been suspected of altering the resultant stem cells so that they are not entirely human. For example, it is suspected that glycosylation patterns and other post-translational modifications may take on characteristics of the feeder cell species.
Thus it would be a great improvement over the state of the art if cell-free methods were developed that support stem cell growth. An even greater improvement would be if stem cells could be grown and harvested using fully characterized, discrete agents wherein as many as possible are synthetic agents. For producing cells suitable for human therapies, it would be a great improvement over the state of the art if methods were developed for culturing the cells that is comprised solely of definable factors. Ideally, the defined components should be free of non-human components. Recombinant proteins, or synthetic components are preferred. Antibodies, including polyclonal, monoclonal, humanized, chimeras or derivatives thereof are especially preferred because their production is highly reproducible, they are robust and they can be readily removed from the harvested cells, for example by affinity depletion using Protein A or Protein G.